Chapter 6 Ag-Ab Interactions:

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Chapter 6 Ag-Ab Interactions
Principles Applications
Fluorescent Ab staining reveals intracellular Ig
Nov 2, 2006
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Chapter 5 Ab Engineering (p. 136 142)
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Human Ab from mice bearing a human artificial
chromosome that includes entire human H- and
L-chain loci
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Derivation of Single-Chain Fragment Variable
(scFv) library
gt 1011
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Phage Display
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Nature of the Ag-Ab interaction Immunological
assays
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4 types of noncovalent forces
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- The interactions of Ab with Ag are through
non-covalent bonds. a. Ionic (or
electrostatic) bond Ionic bonds form
between surfaces of opposite charge.
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b. Hydrogen bond Hydrogen
bonds form between hydrogen atoms and
two other electronegative atoms such
as oxygen and nitrogen.
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c. Van der Waals force Van der
Waals forces occur at very close
ranges between two atoms. Fluctuations in
the electrical charge within electron clouds
can lead to attractive or repulsive forces
between atoms, dependent on the
distance between them.
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d. Hydrophobic bond Hydrophobic
bond is created by the behavior of
hydrophobic subunits in aqueous
environments. These tend to be pushed
together to minimize the instability they
cause in the network of hydrogen- bonded
water molecules.
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Affinity The combined
strength of the noncovalent interactions between
a single Ag-binding site on an Ab and a single
epitope is the affinity of the Ab for that
epitope.
Avidity The strength of multiple interactions
between a multivalent Ab and Ag is called the
avidity. Example secreted pentameric IgM often
has a lower affinity than IgG, but the high
avidity of IgM, resulting from its higher
valence, enables it to bind Ag effectively.
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Cross-reactivity Cross-reactivi
ty occurs if two different Ags share an
identical or very similar epitope.
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Immunological Assays 1. Precipitation
2. Agglutination 3. Radioimmunoassay
4. Enzyme-linked Immunosorbent Assay 5.
Western Blotting 6. Immunofluorescence
7. Flow Cytometry and Fluorescence 8.
Immunoelectron Microscopy 9. Alternatives to
Ag-Ab reactions
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Precipitation lattice formation (??)
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Precipitation is inhibited at Ab excess or Ag
excess
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Precipitation Ab and soluble Ag interacting in
aqueous solution form a visible
precipitate. Agglutination The interaction
between Ab and a particular Ag results in visible
clumping called aglutination.
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Solid-phase radioimmunoassay (RIA)
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Enzyme-linked Immunosorbent Assay (ELISA)
- Indirect ELISA - Sandwich ELISA
- Competitive ELISA -
Chemiluminescence - Elispot assay
Enzymes alkaline phosphatase, horseradish
peroxidase,
?-galactosidase, etc. Substrates
chromogenic (???)
chemiluminescence luxogenic (?????)
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Elispot Assay
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Western Blotting
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Western Blotting
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Immunofluorescence
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Flow Cytometry
FACS Fluoresence-activated cell sorter
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Immunoelectron microscopy
500 nm
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Alternatives to Ag-Ab
reactions Protein A a cell-wall protein from
Staphylococcus aureus (????) Protein G a
cell-wall protein in group C and G Streptococcus
(???) Avidin an egg white protein Streptavidin
a protein made by Streptomyces avidinii -
Protein A protein G can bind to the Fc region
of IgG molecules with high affinity (Kd
10-8 M). - Avidin (or strptavidin) binds biotin
(a vitamin that is essential for fat
synthesis) with Kd 10-15 M.
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Applications Ag Ab Protein A (labeled with
flourochromes,
radioactivity, biotin
or conjugated on
beads) Ag Ab Protein G (labeled with
flourochromes,
radioactivity, or biotin
or conjugated on
beads) Ag Ab (labeled with biotin) avidin or
streptavidin
(conjugated with enzymes,

flourochromes,
radioactivity)
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